Gel electrophoresis is a technique employed primarily in the fields of molecular biology and genetic engineering in which ions are separated by size as they migrate through a gel medium in response to an applied electric field. Various electrophoresis techniques have been employed in the qualitative analysis and separation, recovery and purification of mixtures of macromolecules, particularly mixtures of proteins, nucleic acids, and chromosomes.
Separation of DNA molecules up to about 20,000 base pairs, or 20 kb, in length is routinely achieved by placing DNA in an agarose gel and subjecting the molecules to a constant field. The electric field applies a constant force on the charged DNA molecules and separation is achieved through a length-dependent interaction with the gel matrix. This interaction causes the movement of longer DNA molecules to be retarded to a greater extent than smaller DNA molecules. However, the size of DNA molecules which can be separated by the standard technique is limited since all DNA molecules above about 50,000 base pairs migrate at substantially the same rate.
A variety of modifications of standard electrophoresis techniques have been devised to overcome the size threshold problem. One method of gel electrophoresis capable of separating DNA molecules up to about 2,000 kb in length alternately applies two approximately perpendicular electric fields. One of these fields is uniform and the other is variable. This causes the DNA to migrate along the diagonal in a stair step fashion making right angle turns at each pulse. Length-dependent separation of DNA molecules in the mixture originally applied to the gel is most probably achieved because small molecules turn corners more rapidly than larger molecules. This method results in curved and distorted DNA tracks. See, e.g., Schwartz, D. C. and Cantor, C. R., Cell, 37: 67-75 (1984).
Another improved electrophoresis technique was described by Carle, G. F. and Olson, M. V., Nucleic Acids Research, 12: 5647-5664 (1984). In this method alternate geometries for gel boxes and electrode arrays in an electrophoresis apparatus have been introduced to take advantage of the principle established by Schwartz, et al., supra. According to this method, electrophoresis is conducted by alternately applying two non-uniform electric fields that are approximately orthogonal. The result of using this technique is that the DNA gel tracks are symmetrical in pattern, but the outer tracks are strongly curved, making accurate size comparison difficult.
A more recent attempt to improve the geometry of the DNA tracks involves placing the gel in a vertical position and applying the alternating electric fields at an angle to the plane of the gel. See, e.g., Gardiner, K. et al., Som. Cell Mol. Genet., 12: 185-195 (1986). This approach produces linear DNA tracks by alternating the orientation of applied voltage by 90.degree. . It is the frequency of this alteration which determines the size range of molecules to be resolved. The alternating electric fields are delivered in this method in equivalent pulses and the samples move straight down the lanes directly below the loading wells of the electrophoresis apparatus. However, because of the relatively large cross-sectional area for ion flow, a power supply must be used which is able to deliver relatively high currents at reasonable voltages of approximately 100 to 200 volts. This effectively limits the size of the gel that can be run to approximately 7.times.10 centimeters, or 70 square centimeters in area.
Another electrophoresis technique is described by G. Chu et al, Science, 234: 1582-1586 (1986). This electrophoresis technique is characterized by contour-clamped homogeneous electric fields. The electric fields in this method are arranged along a polygonal contour, and the electric field alternates between two orientations. A recent approach has also been described by Carle, G. F. et al in Science 232: 65-68 (1986). In this method, separation of large DNA molecules is achieved on a standard horizontal gel apparatus by alternately reversing the polarity of a uniform electric field in one dimension. Net forward migration of the DNA molecules is achieved by using a longer pulse time of a fixed voltage in the "forward" direction vs. a shorter pulse time at the same voltage in the "reverse" direction. Thus DNA migrates in the direction of the longer application of uniform voltage. Alternatively, these researchers obtained similar results by employing a constant pulse interval in combination with a higher electric potential in the forward direction and a lower electric potential in the reverse direction, the net DNA migration occurring in the direction of the greater voltage or potential.
There remains a need in the field of molecular biology for additional alternative approaches to electrophoretic separation of large DNA molecules.